Clinical studies targeting aggressive lowering of blood glucose in type 2 diabetes (T2DM) by exogenous insulin failed to show cardiovascular protection, but paradoxically increased cardiovascular mortality. Previously, we demonstrated cerebrovascular insulin resistance (IR) and reduced mitochondria-mediated vasodilation in Zucker obese (ZO) rats with IR compared to lean (ZL) controls. Communication between endoplasmic reticula (ER) and mitochondria play a critical role in metabolic functioning and responses to hypoxia. Tethering of mitochondria to ER by mitofusin 2 (MFN2) creates Ca2+microdomains to aid inter-organelle communication. Overexpression of glucose regulated protein 78 kDa (GRP78), an ER chaperone and a marker of ER stress, is protective by preempting ER Ca2+ leak. Our aim is to demonstrate the adverse impact of IR on ER-mitochondrial interactions. Levels of mitochondrial proteins (MFN2 and voltage dependent anionic channel or VDAC), ER stress marker (GRP78), and endothelial nitric oxide synthase (eNOS) were determined in cerebral arteries from ZO and ZL treated with vehicle or insulin under normoxic or hypoxia-reoxygenation (H-R) conditions. ZO arteries displayed decreased MFN2 and VDAC, elevated GRP78, and increased eNOS monomer/dimer ratio (eNOS uncoupling) at baseline. In ZL arteries, MFN2 and GRP78 proteins were increased under normoxic (1.2±0.1 and 0.97±0.27 respectively; p<0.05 versus vehicle) and decreased under H-R conditions (1.43±0.4 and 0.43±0.02 respectively; p<0.05 versus vehicle) by insulin. In contrast, in ZO arteries, MFN2 and GRP78 proteins were decreased under normoxic (0.69±0.1 and 0.71±0.01 respectively; p<0.05 versus vehicle) and increased under H-R conditions (1.76±0.12 and 1.4±0.07 respectively; p<0.05 versus vehicle) by insulin. Insulin decreased eNOS uncoupling under H-R versus normoxia in ZL, but increased it in ZO arteries. Thus, ZO arteries with IR display impaired mitochondria-ER communication at baseline which was further exacerbated by insulin under H-R leading to increased oxidative vascular injury. Our study provides a mechanistic basis for increased cardiovascular mortality following intensive glucose lowering therapy by high exogenous insulin in patients with IR and T2DM.